Abstract The goal of this proposal is to understand the in vivo mechanism by which STING-IFN? signaling exacerbates pulmonary S. pneumoniae infection. S. pneumoniae infection causes otitis media, pneumonia, septicemia, and meningitis, which kill millions of people worldwide each year especially the elderly and infants. The heavy global burden calls for an enhanced understanding of the host defense mechanism against pneumococci so that novel treatments can be developed. STING (Stimulator of interferon genes) is a stimulator of type I IFN. Current literature supports a protective role for type I IFN in pneumococcal infection. Surprisingly, we first reported that STING-deficient mice are resistant to pneumococcal infection. Consistently, human patients with constitutively activated STING mutations suffer from recurrent pneumococcal infection. Thus, STING mediates host response to pneumococcal infection via a type I IFN independent mechanism. In this proposal, we found surprisingly, that IFN? production is dramatically reduced in lung from infected STING-/- mice at 48hpi. IFN? is an M1 macrophage differentiation cytokine. We found that STING-/- lung macrophages (CD64+CD11B+) have increased Arginase 1 expression and enhanced lung neutrophil efferocytosis indicating an M2-biased phenotype. We further identified lung infiltrating Ly6Chi monocyte as a key player in IFN? production at 48hpi. Mechanistically, we found that heat- kill Streptococcus pneumonia (HKSP) and DNA from dead host cells (self-DNA) can synergistically activate IFN? in Ly6Chi monocyte via IL-12p70/IL-18 synergy. We hypothesize that during the late stage of pneumococcal infection, infiltrating Ly6Chi monocyte, synergistically activated by HKSP and self-DNA, promotes IFN? that drives M1-like, inhibits M2-like macrophage differentiation and exacerbating the disease. Aim1. Determine the cellular mechanisms by which STING stimulates IFN? production during the late-stage of pulmonary pneumococcal infection. Aim2. Uncover a novel molecular mechanism by which bacterial components and self-DNA synergistically activates IFN? production. Achieving these Aims will enhance our understanding of host defense against S pneumoniae infection and pave the way for the development of a new therapeutic strategy for pneumococcal pneumonia.